The invention relates to a new microbiological process for oxidation of methyl groups in aromatic 5- or 6-member ring heterocycles to the corresponding carboxylic acid. The heterocycle has no substituent on the carbon atom adjacent to the methyl group to be oxidized. The heterocycle serves as substrate for the reaction which is performed by microorganisms of the genus Pseudomonas utilizing toluene, xylene or cymene, whose enzymes were previously induced.
2. Background Art
These heterocyclic carboxylic acids, for example, are important intermediate products for the product of pharmaceutical agents. For example, nicotinic acid (3-pyridine carboxylic acid) is an important intermediate product for the production of nicotinic acid amide, which represents a vitamin of the B group and has an essential importance for the nutrition of men and animals [Ullmann, Vol. 19, (1980), p. 603]. 2-Pyrazine carboxylic acid, e.g., is an important intermediate product for the production of the tuberculostatic agent pyrazinamide (2-pyrazine carboxylic acid amide), [Roemps Chemie Lexikon, Vol. 5, (1987), 3411]. 4-Thiazole carboxylic acid serves for the production of thiabendazole, a highly effective antihelminthic (helminthicide), which in turn is a starting material for other newer antihelminthic agents, such as, cambendazole [Ullmann, Vol. 23, (1980), p. 146]. 2-Thiophene carboxylic acid has an antiallergic effect [Ullmann, Vol. 23, (1980), p. 219].
In-depth investigations for the oxidation of methyl groups so far have been conducted with aromatic hydrocarbons.
The production of carboxylic acids by the microbial oxidation of methylated aromatic compounds was exhaustively described in the works of Raymond et al. [Raymond et al., Process Biochem., (1969), pp. 71 to 74]. U.S. Pat. No. 3,393,289 describes a process for the biochemical oxidation of methyl groups in aromatic hydrocarbons with a gram-positive microorganism strain of the genus Nocardia. Drawbacks of these processes are that, for example, in the methyl group oxidation of aromatic hydrocarbons the benzene ring of the corresponding acid is cleaved off.
In regards to the use of Pseudomonas putida ATCC 33015, it is known that the biochemical oxidation of the methyl group from toluene to benzoic acid takes place in three steps. By the action of toluene monoxygenase first benzyl alcohol results, which then in two other steps, catalyzed by an alcohol dehydrogenase and aldehyde dehydrogenase, is reacted to the acid. In this strain, both the Xyl gene, which codes for enzymes of the xylene degradation, and the genes, which are responsible for the regulation of the Xyl gene, lie on the plasmid pWWO. This archetypal Tol plasmid has already been thoroughly studied in a molecular biological manner [Harayama et al., J. Bacteriol. 171, 1989), pp. 5048 to 5055; Burlage et al., Appl. Environ Microbiol. 55, (1989), pp. 1323 to 1328].
Likewise, microbiological processes for the oxidation of methyl groups of an N-heterocycle are also known from the literature. According to Soviet Union Patent No. 417,468, 2-methyl pyridine is oxidized with a gram-positive microorganism strain of the genus Nocardia to the corresponding acid.
Soviet Union Patent No. 228,688 describes a microbiological process for the production of nicotinic acid from 3-methyl pyridine with a gram-positive microorganism of the genus Mycobacterium. A microbiological process for the production of nicotinic acid with gram-positive bacteria of the genus Nocardia is known from Soviet Union Patent No. 302,341.
The drawbacks of the methyl groups oxidation of N-heterocycles with gram-positive bacteria are that, with such alkane-utilizing bacteria the mixture ratio of the alkane to the substance to be oxidized has to be adjusted exactly to achieve a biotransformation and that no biotransformation of the substrate occurs in the absence of the alkane, i.e., the alkane used for the induction, always has to be present, also in the reaction of the substrate. By comparative tests with the gram-positive bacterium Nocardia and applicants' gram-negative Pseudomonas, it was clearly shown that using Nocardia even in the presence of an alkane, such as, dodecane, 3-methyl pyridine could not be oxidized to nicotinic acid.
In addition, U.S. Pat. No. 4,859,592 describes a process for the production of picolinic acid with Pseudomonas putida by an alkyl-substituted aromatic hydrocarbon being formed in the presence of molecular oxygen in a first step by a dioxygenase into a 2-hydroxy muconic acid semialdehyde, and then the latter being reacted in a second step with ammonia or a primary amine to 2-picolinic acid. The drawback of such process is that the corresponding picolinic acid is formed only in the second step by the reaction of the 2-hydroxy muconic acid semialdehyde with ammonia.